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Download fileA Thymine Isostere in the Templating Position Disrupts Assembly of the Closed DNA Polymerase β Ternary Complex
journal contribution
posted on 2005-11-22, 00:00 authored by Thomas W. Kirby, Eugene F. DeRose, William A. Beard, Samuel H. Wilson, Robert E. LondonThe high fidelity of the DNA polymerization process is critically important for the stability of
the cellular genome. The role of template and incoming nucleotide base pairing in polymerase fidelity
has recently been explored by the use of nucleotide isosteres, which preserve the steric but not the electronic
properties of the corresponding bases. The DNA repair enzyme, DNA polymerase β (Pol β), is among
the most discriminating, being inactive when the thymine isostere difluorotoluene (DFT) is present in the
templating base position. To explore the physical basis for this inactivity, we have performed NMR studies
on [methyl-13C]methionine-labeled Pol β complexed with double-hairpin DNA, used to model the gapped
nucleotide substrate, and having either a thymine or a DFT isostere at the templating base position. The
six methionine residues distributed throughout the enzyme provide useful conformational probes of the
lyase and polymerase domains and subdomains. Analysis of the proton shift of Met282 that results from
formation of an abortive Pol β−gapped DNA−dATP complex is consistent with an open to closed
conformational change of the enzyme predicted from crystal structures. In contrast, the same resonance
is nearly unshifted when a ternary complex is formed from dATP and gapped DNA in which a DFT
isostere replaces thymine at the templating base position. Alternatively, the resonances of Met191 and
Met155, located in the catalytic subdomain, show perturbations upon formation of the abortive ternary
complex, which are qualitatively similar, but significantly weaker, than the changes observed when thymine
is present at the templating base position. The changes in the Met155 and Met191 methyl resonances are
in fact more similar to those observed in the binary Pol β−dATP complex. These studies demonstrate
that the block in catalysis is directly related to the absence of the set of conformational transitions that
include the “open” to “closed” transition monitored by Met282.